The goal of this project is to use targeted gene disruption of MEK kinase 2 (MEKK2) to determine its role in regulation of signaling pathways and of gene expression in hematopoietic cells. Preliminary date has shown that loss of MEKK2 in ES cell derived mast cells results in loss JNK activation in response to c-Kit and FcepsilonRI ligation and there is significantly decreased production of selected cytokines in the stimulated cells. In T cells with activation by T cell/antigen presenting cell conjugate formation, MEKK2 rapidly translocates to the conjugate interface. Transfected kinase inactive MEKK2 prevents conjugate formation and slightly inhibits IL-4 production. Specific aims of this project are to generate MEKK2 knockout mice, to determine the knockout phenotype, and to collect mast cells and T cells from the mice to study effects of loss of MEKK2 on signaling pathways, cell proliferation, and cytokine production. Novel techniques, such as in vitro differentiation of ES cells and microchip DNA array technology, will be used. Results of these studies will have an impact on the understanding of MEKK2's role in signaling pathways as well as its role in control of cytokine production and immune function. They could also have implications in the field of cancer research, since transfected MEKK2 in T cells can affect rate of cell cycling. Ultimately, these studies may lead to the development of novel therapies targeted at influencing MEKK2 activity to treat cancers or to modulate immune function. As a pediatric hematologist, oncologist, this project will be invaluable in teaching me research methods in the areas of cell signaling, control of cell growth, and immunology, all areas intimately involved with my field. My long term goal is to apply this growth, and immunology, all areas intimately involved with my field. My long term goal is to apply this knowledge to gain a better understanding of cellular events leading to dysregulation of signaling pathways and resulting in cancer in children, so that effective, targeted therapies can be devised. Gary Johnson's lab is an ideal environment in which to learn these skills.